Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.

Neutrophils are mobilized to the vascular wall during vessel inflammation. Published data are conflicting on phagocytic nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation during the hypertensive state, and the capacity of angiotensin II (Ang II) to modulate the intracellular redox status has not been analyzed in neutrophils. We here describe that Ang II highly stimulates endogenous and extracellular O 2 ؊ production in these cells, consistent with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47 phox , and p67 phox . The Ang II-dependent O 2 ؊ production was suppressed by specific inhibitors of AT1 receptors, of the p38MAPK and ERK1/2 pathways, and of flavin oxidases. Furthermore, Ang II induced a robust phosphorylation of p38MAPK, ERK1/2, and JNK1/2 (particularly JNK2), which was hindered by inhibitors of NADPH oxidase, tyrosine kinases, and ROS scavengers. Ang II increased cytosolic Ca 2؉ levels-released mainly from calcium stores-enhanced the syn- IntroductionAngiotensin II (Ang II), the main peptide hormone of the reninangiotensin system, induces leukocyte recruitment to the vessel wall, which constitutes a hallmark of the early stages of atherosclerosis and several hypertensive diseases. 1 In addition, it plays a regulatory role on blood pressure and circulation volume and on the proliferation of vascular smooth muscle cells. 2 Ang II acts through high-affinity cell surface receptors (AT1), which are linked to pathways classically associated with G-protein-coupled and tyrosine-kinase-mediated responses. 3 Although most studies on Ang II have been carried out in smooth muscle and endothelial cells, experimental evidence has been obtained on its effects on circulating cells. AT1 receptors for Ang II have been found recently in circulating neutrophils 4 and human peripheral monocytes, 5 and Ang II-induced cell activation in the latter has been reported. 6 In this context, the migration of leukocytes from blood to sites of inflammation and their adhesion to endothelial cells are primary events taking place during the acute inflammatory response and the pathogenesis of vascular diseases. 7 Because chronic inflammation of vessel walls is a hallmark of atherosclerosis, 8 and reactive oxygen species (ROS) such as superoxide anion (O 2 Ϫ ) and H 2 O 2 constitute the main intermediary molecules responsible for inflammation, 9 a link between atherosclerosis and ROS production has been postulated. 10 Nicotinamideadenine dinucleotide phosphate (NADPH) oxidase of phagocytes catalyzes the reduction of oxygen to O 2 Ϫ . In resting cells this enzyme is inactive, and its components are distributed between the cytosol and the membrane of secretory vesicles. When phagocytic cells are activated, the enzyme's cytosolic components associate to membrane-bound components and assemble into catalytically active NADPH oxidase. 11 It has been reported that Ang II-induced hypertrophy of vascular smooth muscle cells is mediated by both O 2 Ϫ and H 2 O 2 . 12 In addition, ROS ha...

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS-PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

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The histophagous ciliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus and sea bass Dicentrarchus labrax. The present study screened 52 candidate antiprotozoals for activity against this pathogen in vitro. Of these compounds, 14 were effective (i.e. killed all ciliates within a 24 h assay period). In descending order of efficacy (minimum lethal concentration 100 to 0.8 ppm), these were niclosamide, oxyclozanide, bithionol sulfoxide, toltrazuril, N-(2'-hydroxy-5'-chloro-benzoyl) 2-chloro-4-nitroaniline, furaltadone, doxycycline hyclate, formalin, albendazole, carnidazole, pyrimethamine, quinacrine hydrochloride and quinine sulfate. Administration in filtered seawater rather than phosphate-buffered saline inactivated doxycycline hyclate and albendazole, and markedly reduced that of bithionol sulfoxide and toltrazuril, suggesting that these compounds may not be effective in bath administration. In view of these findings, we discuss the potential utility of chemotherapy as a strategy for the control of scuticociliatosis in farmed turbot and sea bass. KEY WORDS: Turbot · Scuticociliatosis · Philasterides dicentrarchi · Chemotherapy · In vitro assayResale or republication not permitted without written consent of the publisher Dis Aquat Org 49: 191-197, 2002 192 pensions of powder compounds (tablets were previously powdered in a mortar) were prepared in distilled water or dimethylsulfoxide (DMSO; Sigma Chemical). The resulting stocks, or commercially purchased solutions, were diluted in physiological phosphatebuffered saline (PBS; pH 7.2) or 0.2 µm-filtered seawater (salinity 28 ‰) to the final concentrations used in the screening (see following subsection). Tests performed in filtered seawater allow automatic exclusion of the possibility of partial or total inactivation of the test substance under marine conditions. A substance found to be effective in vitro by this procedure can thus be expected to be effective in bath administration to infected fish. Determination of lethal activity. Ciliates in the late exponential phase or early plateau phase of culture were concentrated by centrifugation at 650 × g for 5 min and then resuspended in PBS or filtered seawater. After counting in a haemocytometer, 10 µl of ciliate suspension containing 10 4 ciliates were added to each well of 96-well microtitre polystyrene plates containing 90µl well -1 of the candidate antiprotozoal at the required dose in PBS or filtered seawater. Final doses tested were: (1) 250, 125, 62, 31, 16, 8.4, and 2 ppm for formalin, and (2) 100, 50, 25, 12.5, 6.2, 3.1, 1.5 and 0.8 ppm for the remaining test substances. Each determination was performed in duplicate. Wells containing ciliates in assay solution (PBS or filtered seawater) without chemicals were also assayed as negative controls. To rule out possible effects of the solvent in DMSO-dissolved compounds, duplicate wells with PBS or filtered seawater containing the highest concentration of DMSO used (up to 2.5%) were also included. Pl...

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This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.